All publications cited in the present application are incorporated in their entirety by reference.
1. Field of the Invention
The present application relates to nucleic acids encoding β-secretase (BACE) polypeptides, methods for making BACE polypeptides, methods for growing crystals of BACE, crystalline BACE, the three-dimensional structure of BACE, and the use of the crystalline forms to identify ligands, such as antagonists, that bind to BACE.
2. Invention Background
Alzheimer's disease (AD) is a neurodegenerative disease characterized by neuronal loss due to the extracellular accumulation of amyloid plaques and intracellular accumulation of neurofibrillary tangles in the brain (reviewed by Selkoe, D.J. (1999) Nature 399: A23-31). Two major components of amyloid plaques are small peptide fragments Aβ40 and Aβ42, which are generated from cleavage of the membrane-anchored amyloid precursor protein (APP) by the proteolytic activity of β- and γ-secretases. APP is a type I integral membrane protein containing the Aβ segment, which begins at D672 in the longest isoform and spans the boundary of the exocytoplasmic region (28 amino acids) and the transmembrane domain (12-14 amino acids). The γ-secretase activity cleaves APP within the transmembrane domain to produce the carboxy-terminal end of Aβ polypeptide. The β-secretase activity (aspartic protease activity), identified in a protein that is known as “mamapsin 2”, “human β-site APP-cleaving enzyme” or “BACE”, and “Asp 2”, cleaves APP on the extracellular side of the membrane to produce the amino-terminal end of Aβ. (Vassar, R. et al., (1999) Science 286,735, Sinha, S. et al., (1999) Nature 402,537, Yan, R. et al., (1999) Nature 402,522, Hussain, I. et al., (1999) Mol. Cell Neurosci. 14, 419 and Lin, X. (2000) et al., Proc. Natl. Acad. Sci. USA 97,1456. Another enzyme, known as α-secretase, cleaves APP at a position within the Aβ sequence to produce a soluble APPα (Esch et al., (1990) Science 248: 1122-1124).
During the course of AD, Aβ polypeptide accumulates extracellularly in the brain, and forms large, insoluble amyloid fibrils that elicit both cytotoxic and inflammatory responses. Thus, BACE and γ-secretase proteases are targets for potential inhibitor drugs (antagonists) against AD. Because it was discovered that BACE activity is the rate-limiting step in Aβ production in vivo (Sinha and Lieberburg, (1999) Proc. Natl. Acad. Sci. USA 96: 11049), BACE has become a prime target for the development of inhibitors (antagonists) to treat AD.
The BACE gene encodes a 501 residue polypeptide having, from N- to C-terminus, an N-terminal signal sequence of 21 amino acids; a pro-protein domain of 22 to 45 residues, which is proteolytically removed by furin to generate mature β-secretase (Creemers, J.W., et al. (2001) J. Biol. Chem. 276: 4211-4217; Bennet, B.D., et al. (2000) J. Biol. Chem. 275: 37712-37717); a protease (catalytic) domain; a connecting strand, an integral membrane (transmembrane) domain of about 17 amino acids; and a short cytosolic C-terminal tail of 24 amino acids (Vassar et al., supra). Sequence analyses indicate that BACE belongs to a subfamily of membrane-bound and soluble proteases, and contains a classic consensus active site motif found in aspartyl proteases (D T/S G T/S) at positions 93 to 96 and 289 to 292. The entire BACE sequence displays only mild homology with known aspartyl proteases, approximately 30% identity and 37% similarity with members of the mammalian pepsin family, with the highest homology found in the central portion of the extracellular domain.
Accurate information regarding the three-dimensional structure of β-secretase is helpful in the design and identification of ligands, particularly inhibitors (antagonists), of BACE, and in the enzymatic characterization of the enzyme. This information may be provided using crystals of the protein in X-ray crytallographic analysis.
Crystallization of a protein is a very time consuming and complex process. Crystallization of a protein requires a precise set of reagents and reaction conditions that promote the growth of crystallized protein. For example, specific amounts of protein, buffer, precipitating agent and salt, at a precise temperature, are required to produce X-ray diffraction quality crystals. There are an infinite number of combinations of the above reagents and reaction conditions. Therefore, the number of different combinations that can be tested is limited by the amount of protein that can be produced. Because the precise set of conditions that will produce crystals can not be predicted, one is more likely to discover crystals as more reagents and reaction conditions are tested. As a result, effective crystallization requires a large amount of refolded protein, typically milligram quantities. This is problematic because current methods for expressing BACE in E. coli provide low yields of unfolded protein. In addition, large amounts of unfolded protein are required to optimize the protein's refolding procedures. Thus, there is a need for nucleic acids encoding BACE that are optimized for E. coli expression, which utilize codons that are preferred by E. coli, to produce large quantities of BACE to both discover optimal refolding conditions and so that many different combinations of the above reagents and reaction conditions may be tested in order to optimize the crystallization conditions for BACE.
A crystal form of β-secretase complexed to an inhibitor is described in Hong et al., (2000) Science 290:150-153. In addition, several international applications published under the Patent Cooperation Treaty, international publication numbers WO 02/25276 A1, WO 01/00663 A2 and WO 01/00665 A2, provide crystal forms of BACE complexed to an inhibitor. Knowledge of the structure of a protein in both the uncomplexed and complexed forms allows one to determine how the three-dimensional structure of the protein changes upon binding to a ligand. This aids in structure based drug design because it provides more information regarding how a particular ligand may be altered to increase its binding to the protein. Thus, there is a need for crystals of β-secretase which have similar structure and activity to that of native BACE, and which can be produced in the uncomplexed form.